There is an urgent need for the development of novel approaches to study fungal pathogenesis. However, some virulence traits are induced only in the host and therefore the identification of these virulence traits may require detection in vivo. The main objective of this proposal is the development and implementation of whole animal Caenorhabditis elegans-based, genome-wide assays to study fungal virulence. The C. elegans model provides an unambiguous assay endpoint in the survival/death of the worms, allows the use of robots for filling assay plates and permits automated readouts using plate readers and imaging microscopes. The studies detailed in this proposal are expected to develop high-throughput pathogenesis assays that are based on the C. elegans systems of fungal pathogenesis, and implement these assays for the identification of virulence traits. These studies will expand the C. elegans models of fungal pathogenesis and identify genes of fundamental relevance to pathogenesis, independent of the model system used. There are two Aims in this proposal: 1. Develop a large collection of random Cryptococcus neoformans mutants and implement C. elegans assays to identify virulence associated genes, and, 2. Implement whole animal C. elegans assays to identify virulence traits associated with Candida albicans and Candida glabrata infection. Based on our previous C. elegans assays, including C. neoformans assay that utilized a library similar to the one proposed in Aim 1 and our studies of the Candida libraries, we expect to identify a total of 800-1,000 hypovirulent mutants. Based on our experience with this model, we anticipate that we shall be able to identify the disrupted genes in the majority of these mutants and most of these genes will be involved in mammalian infection. Importantly, virulence genes will be confirmed, studied and prioritized and all phenotypic data from the mutants will be accessible via a public database and all strains will be publicly available. We anticipate that these collections will be an important resource for workers that study fungal pathogenesis. C. elegans is an exciting, ethically expedient, rapid and inexpensive substitute host to study fungal pathogenesis. C. elegans assays minimize mammalian suffering and enable the study of fungal pathogenesis in a system where both the pathogen and the host can be genetically manipulated. We anticipate that this model system will expand our understanding of evolutionarily conserved elements of fungal pathogenesis and provide a new and exciting way to study fungal virulence. Moreover, these studies will serve as a paradigm of future studies in other pathogens that the infectious process can be studied in vivo in genome-wide scale utilizing a relatively simple model host. PUBLIC HEALTH RELEVANCE: There is an urgent need for the development of new approaches to study fungal pathogenesis. The main objective of this proposal is the development of high throughput, whole animal Caenorhabditis elegans-based assays that can be used to study fungal pathogenesis. This model can minimize mammalian suffering and help identify some basic aspects of fungal pathogenesis and host response.